Cryopreservation of articular cartilage

ABSTRACT

The invention relates generally to methods and compositions for the cryopreservation and/or vitrification of tissue including articular cartilage and the preparation of said tissue for clinical or research use, including but not limited to joint replacement and the treatment and prevention of osteoarthritis.

FIELD

Cryopreservation of biological tissues, including articular cartilage.

BACKGROUND

There is much need for the preservation of cells and tissues, forinstance, in the preservation, research and transplantation of articularcartilage or joints. Certain conditions can benefit from whole orpartial joint replacement, such as osteoarthritis. Furthermore,osteochondral allografting of large joint defects (due to injury ordisease) can maintain joint function and decrease the incidence ofosteoarthritis. Unfortunately, the use of such procedures is limited bythe availability of appropriate tissue.

Cryobiology is the study of the effects of extremely low temperatures onbiological systems, with a major application being the storage of cellsand tissues for research and transplantation to treat injury anddisease. Cryopreservation is currently the only method available topreserve long-term function and viability of mammalian cells and tissue.Many tissue types have eluded successful cryopreservation.

Currently, there are no effective cryopreservation techniques forarticular cartilage (AC). Vitrification is one potential method butcurrent processes are unsuccessful due to inadequate cryoprotectantagents (CPAs) permeation and toxicity of these CPAs.

SUMMARY

The inventors have developed a method for cryopreserving tissue, such asAC, for transplantation or research.

In an embodiment, there is provided a method for cryopreservingarticular cartilage using more than one cryopreserving agent (CPA), themethod comprising permeating a sample of articular cartilage with asequence of at least two different CPAs comprising a first CPA and asecond CPA, the second CPA being permeated into the sample afterpermeating the sample with the first CPA, to form combined CPAs having aconcentration distribution within the sample, the concentrationdistribution of the combined CPAs being selected so that upon cooling ofthe sample, the combined CPAs vitrify and cryopreserve the sample. Thesequence may include other CPAs, for example a total of four, five, sixor seven or more CPAs. The CPAs may be selected from the groupcomprising dimethyl sulfoxide (D), ethylene glycol (EG), propyleneglycol (PG), glycerol, (G) formamide (F), methanol (Me), ethanol (Et).Exemplary CPA sequences include D-G-PG-EG, G-EG-D-F, EG-G-D-PG,EG-G-F-D, and G-D-EG-F. Other embodiments are found in the claims, whichare incorporated here by reference.

In another embodiment, there is provided a kit of compositions for usein the preservation of a sample of articular cartilage. In oneembodiment said composition comprises two or more CPAs, or mixturesthereof. The kit may include instructions on how to preserve the sample.

Other features and advantages of the present invention will becomeapparent from the following detailed description and accompanyingdrawings. It should be understood, however, that the detaileddescription and the specific examples while indicating preferredembodiments of the invention are given by way of illustration only,since various changes and modifications of what is disclosed areintended to be covered by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1 is a flow chart illustrating one embodiment of a method for thecryopreservation of cartilage.

FIG. 2 is a graph showing the increase in minimum cartilage DMSOconcentration (minimum located at the bone-cartilage interface) incartilage with 2 mm thickness with DMSO concentration of 3 M in theexternal bath for 180 minutes.

GLOSSARY

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the terms “comprising,” “including,” and “such as” areused in their open and non-limiting sense.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about”. The term “about” means plus or minus 10%, and includes anyrange up to and including 10%, of the number to which reference is beingmade.

Further, it is to be understood that “a,” “an,” and “the” include theplural reference unless the content clearly dictates otherwise. Forexample, reference to “a compound” includes a mixture of two or morecompounds. Thus, the phrase “a CPA”, as used herein can also mean “oneor more CPAs” or “at least one CPA” unless the context dictatesotherwise.

A “beneficial effect” refers to favourable pharmacological and/ortherapeutic effects, and/or improved pharmacokinetic properties andbiological activity of at least one tissue, such as AC. A beneficialeffect or sustained beneficial effect may manifest as decreased or node-vitrification of tissue during the cryopreservation process and/or indesired or improved tissue or cell viability. In aspects of theinvention, for instance in tissue transplantation, beneficial effectsinclude but are not limited to decreased disease progression, decreasedor alleviated disease symptoms, increased survival, or elimination orpartial elimination of a condition and/or disease.

The structure of agents identified by generic or trade names herein maybe taken from the standard compendium “The Merck Index” or fromdatabases such as PubMed(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi), and patent databases(http://www.uspto.gov/patft/index.html;http://patents1.ic.gc.ca/intro-e.html;http://register.epoline.org/espacenet/ep/en/srch-reg.htm). A personskilled in the art using these references is fully enabled to identify,manufacture and test the indications and properties in standard testmodels, both in vitro and in vivo.

“Condition(s) and/or disease(s)” refers to one or more pathologicalsymptoms or syndromes for which the tissues or cells preserved hereinprovide a beneficial effect or therapeutic effect. Examples ofconditions and/or diseases include but are not limited toosteoarthritis, tumours, avascular necrosis or traumatic joint defects.

“Vitrification” as used herein refers to the formation of an amorphoussolid from an aqueous solution without significant crystal formationthat usually requires a combination of high concentrations of CPAsand/or rapid cooling.

“De-vitrification” as used herein refers to the formation of icecrystals in a fluid upon re-warming from a vitrified state.

“Cryopreservation” as used herein refers to the process of cooling cellsand tissues to ultra-low temperatures at which biochemical processes aresignificantly slowed.

Abbreviations used include dimethyl sulfoxide (DMSO; D), ethylene glycol(EG), propylene glycol (PG), glycerol (gly; G), formamide (form; F),methanol (Me), ethanol (Et), chondroitin sulphate (CS; cond sulp, condsulf), hyaluronic acid (HA), hours (hr), minutes (min), standarddeviation (std dev), average (ave; avg), molar (M).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

There is disclosed a method for cryopreserving articular cartilage usingmore than one cryopreserving agent (CPA). The method comprisespermeating a sample of articular cartilage with a sequence of at leasttwo different CPAs comprising a first CPA and a second CPA, the secondCPA being permeated into the sample after permeating the sample with thefirst CPA, to form combined CPAs having a concentration distributionwithin the sample, the concentration distribution of the combined CPAsbeing selected so that upon cooling of the sample, the combined CPAsvitrify and cryopreserve the sample. By using different CPAs permeatedinto the sample sequentially, a lower toxicity is obtained than would beexpected for a given combination of CPAs. That is, toxicity is notadditive. The sequence may include other CPAs, for example a total offour CPAs. The CPAs may be selected from the group comprising (but notlimited to) dimethyl sulfoxide (D), ethylene glycol (EG), propyleneglycol (PG), glycerol (G), formamide (F), methanol (Me) and ethanol(Et). Exemplary CPA sequences include D-G-PG-EG, G-EG-D-F, EG-G-D-PG,EG-G-F-D, and G-D-EG-F. Variations in cryopreservation success may stillbe obtained, however, due to sample differences, changes in toxicity dueto temperature variation and interaction of the CPAs used.

In considering the results of following the disclosed methods,sufficient success is obtained from any part of the sample survivingcryopreservation and warming since the threshold to beat is deadcartilage with no surviving chondrocytes. In the normal and expecteduse, the sample is typically taken from a human donor, either alive ordead. The cryopreserved sample may be used for a variety of purposes,such as study, or implantation into a different person or animal, butwill not be returned to the body from which it was taken. Addition ofchondroitin sulphate or hyaluronic acid to one or more of the CPAs mayalso be part of a cryopreservation method. Based on the examples of useof chondroitin sulphate shown below, chondroitin sulphate may be addedto any or all of the solutions in an amount of, for example, from 0.1 to10 mg/ml of the containing solution. Based on the example of use ofhyaluronic acid shown below, hyaluronic acid may be added to any or allof the solutions in an amount of, for example, from 0.1 to 10 mg/ml ofthe containing solution. A first CPA may be permeated into tissue incombination with other CPAs provided there is a separate and subsequentpermeation with at least a further CPA or combination of CPAs having adifferent composition than the first CPA or combination of CPAs.

In permeating the sample with a sequence of CPAs to obtain aconcentration distribution of combined CPAs within a sample that permitsvitrification of the combined CPAs and cryopreservation of the tissue,the CPAs should be permeated at suitable concentrations, times andtemperatures. In accordance with normal cryopreservation techniques, thetemperature of application of a sequence of CPAs normally does notincrease from CPA to CPA but stays the same or decreases from one CPAapplication to another. In addition, and this would be appreciated by aperson of average skill in the art, the temperature of the CPA andtissue should not be below the freezing point of the CPA or the tissue.The concentration and total time of exposure and temperature history ofthe CPA should be at a toxicity that is dependent on the CPA and that isnot excessively toxic to the sample. CPA toxicities are known ordeterminable and thus application of the CPAs at a suitable toxicity iswithin the skill of a person of average skill in the art. The timeallowed for permeation may be calculated following published permeationalgorithms to achieve a level of permeation that enables vitrificationof the combined CPAs and subsequent cryopreservation of the sample. Inaddition, methods of calculating desired permeation are also disclosedhere.

The basic approach to permeating the sample of cartilage with a sequenceof CPAs is to start with cartilage that has no CPA inside and put CPA 1plus buffered saline or suitable media outside. Water and CPA then move,with the CPA permeating the cartilage, and the end result, approachingequilibrium, is that the cartilage now contains some CPA 1. Next, it isdesired to add CPA 2, a different CPA from CPA 1, to the cartilage. CPA1 should stay in the cartilage while CPA 2 is added. Hence, the CPA 2should be added with CPA 1 at the concentration of CPA 1 already in thecartilage. The CPA 2 is added at a concentration that allows the CPA 2to permeate the cartilage to a desired level in a reasonable amount oftime without being too toxic. Thus, the preferred amount of CPA 1present in the permeating CPA 2 solution is close to the same level asthe concentration of CPA 1 already in the sample when CPA 2 is added. Itis possible to have the amount of CPA 1 in the cartilage sample at thetime of beginning diffusion of CPA 2 deviate from the desired finalconcentration, but the greater this deviation differs (higher or lower)from the desired final concentration, the more complicated the processbecomes possibly without a corresponding benefit. In addition, if theCPA 1 in the cartilage is raised to a higher level than the ultimatedesired concentration then the sample cartilage is exposed tounnecessarily high toxicity. A similar principle applies to the additionof any succeeding CPA, call it CPA n, where the amount of preceding CPAadded with CPA n is preferably, but not always necessarily, at theconcentration of the preceding CPA in the sample cartilage.

Mathematical models of freezing points may be used to determine themaximum amount that the temperature could be lowered in the next step.In one embodiment this could be calculation of the liquidus (freezingpoint) of the solution at the point in the tissue with the least amountof CPA. In another embodiment this could be calculation of the liquidus(freezing point) of the solution corresponding to the average amount ofCPA in the tissue. In another embodiment this could be calculation ofthe liquidus (freezing point) of the solution corresponding to theminimum amount of CPA throughout the tissue. In one aspect the freezingpoint of specific solutions may be determined by any standard methodsuch as differential scanning calorimetry (DSC) or differential thermalanalysis (DTA). In another aspect the freezing point of the solution isdetermined by any one of a number of published models of freezing pointof single or multi-solute aqueous solutions including the osmotic virialequation, or by manual determination with constant monitoring oftemperature during the cooling process.

The guiding principles for arriving at a particular example of theinvention include relative toxicity of CPAs at room temperature andother temperatures, computed permeation times for CPAs, computedfreezing points after permeation, and a computation of whether or not aparticular combination at specific concentrations will vitrify.

For example, in one embodiment, the tissue to be vitrified is added to aspecific concentration of a low toxicity CPA (for example, EG) at 0° C.for a pre-specified time. After the pre-specified time (sufficient toachieve the minimum pre-specified permeation throughout the tissue), thetissue will be moved to another solution that contains two CPAs at alower temperature (just above the freezing point of the expectedstarting CPA concentration within the tissue determined by amathematical model). The determination of this second solutionpreferably uses combined CPAs that minimize toxicity, improvepermeation, and enhance vitrification. Once again, the tissue will beleft in this solution for a length of time to allow permeation to aminimum desired concentration. This can be repeated two or more timesuntil a high enough concentration of all the different CPAs is achievedto vitrify the solution and effectively cryopreserve the tissue. In oneembodiment it is repeated two times. In another embodiment it isrepeated more than two times.

The method may use statistical assessment of relative toxicity of CPAsand/or mathematical models of permeation kinetics to determineparameters of addition/dilution of multiple CPAs in a step-wise mannerat progressively lower temperatures resulting in progressively higherCPA concentrations until a sufficient concentration to vitrify isachieved. Exposure times can be mathematically determined for specifictissue thickness to optimize permeation while minimizing toxicity. Inone aspect, the individual CPAs are added at different temperatures sothat the ratios of the CPA concentrations changes throughout theprotocol.

In a further embodiment, a method for cryopreserving articular cartilageusing more than one cryopreserving agent comprises:

-   -   (i) obtaining an articular cartilage sample;    -   (ii) adding one CPA first at a temperature above the freezing        point of the native tissue and CPA bathing solution for a        sufficient period of time to obtain a desired degree of CPA        tissue permeation;    -   (iii) moving the tissue to another solution that contains at        least one or more

CPAs at the same or lower temperature then the temperature in step“(ii)”, but higher than the freezing point of the solution and tissue instep (ii), for a sufficient period of time to obtain a desired degree ofCPA tissue permeation;

-   -   (iv) repeating step (iii) with different CPAs at the same or        lower temperatures than previously used but higher than the        solution and tissue freezing point, until a high enough        concentration of all the different CPAs in the tissue is        achieved to vitrify the solution and effectively cryopreserve        the tissue. In one embodiment, step (iii) is repeated two times.        In another embodiment, step (iii) is repeated more than two        times.

In one embodiment, the articular cartilage sample is obtained from anymammal including but not limited to humans, preferably human. It isnoted that skeletally mature pig knee joints are slightly smaller thanhuman knee joints but that the cartilage thickness is similar betweenthe two, so for this reason, it is considered to be one of the bestmodels for cartilage transplantation procedures. It is noted thatpersons skilled in the art are familiar with various transplantationtechniques, for instance a suitable osteochondral allografting surgicaltechnique.

The thickness of the articular cartilage sample may be 1-6 mm, above 1mm, or between 2 to 6 mm for example. The toxicity of the CPA may bedetermined by membrane integrity assays of slices taken from wholedowels post treatment or from published data for example. A dual staintechnique may be used whereby intact cells will fluoresce a green colourwhile those with damaged membranes will fluoresce a red colour asdescribed below. In one aspect, CPAs of similar toxicity could beadministered in combination. In one aspect, they have differentpermeation kinetics, in another aspect they have similar permeationkinetics.

In one embodiment, the CPAs may be added in order of increasingtoxicity. In another embodiment the CPAs applied subsequently to thefirst CPA have similar toxicity and can be administered in combination.In another embodiment, the CPAs may be administered based on permeationkinetics.

In one embodiment, certain CPAs may interact and result in differenttoxicity or permeation kinetics than if administered alone oradministered without any subsequent or previous tissue treatment withother CPAs or compounds.

In one embodiment, the sufficient time for tissue permeation, is thetime for sufficient permeation to obtain vitrification but no or minimalde-vitrification. In another embodiment it is full permeation of thetissue with the respective CPA or CPA solution.

In one method the relative toxicity of a CPA or combination thereof isdetermined by administering the CPA to a tissue sample or individualcells and then determining cell or tissue viability using knowntechniques, such as cell staining with Syto 13 and ethidium bromide,wherein intact cells are green and disrupted cells are red. The degreeof cell viability can be obtained by counting the respective cells overa specified area. This can be done over various different time pointsand at different temperatures. Other methods include assessment ofmetabolic activity using a test such as WST-1 that measuresmitochondrial activity.

In one method permeation kinetics of a CPA or combination thereof aredetermined by measurement of the amount of CPA that has diffused into aknown quantity of tissue after specified periods of time and at specifictemperatures. Another method would be to use magnetic resonance imaging.

The degree of success of the vitrification or cryopreservation techniquecan be assessed by determining cell or tissue viability as previouslydescribed from a portion of the tissue sample to be used or a controlsample. Positive and negative controls or both can be used. The degreeof any de-vitrification can be observed visually (e.g. formation of icecrystals upon rewarming) by loss of glass clarity and the formation ofcracks.

The disclosed methods may be used for preparing cryopreserved tissue forclinical or research use. In one embodiment, said use includes a step orsteps for removing CPA from the tissue to a minimized toxicity level. Inone embodiment, it involves removing all or essentially all of the CPAfrom the tissue, in preparation for research or transplantation. Inanother embodiment, there is provided a method for warming the tissue tothe desired temperature for use. In one embodiment, the said methodsused preferably obtain the desired degree of tissue or cell viabilityfor the intended use.

There is also provided a kit comprising one or more solutions containingin each solution one or more CPAs and optionally instructions forvitrification of tissue. A kit may comprise a package which houses acontainer which contains a composition of CPAs and also housesinstructions for cryopreserving the tissue or articular cartilage as perthe method of the invention. In one embodiment, the instructions furtherinclude instructions for preparing the tissue or articular cartilage fortransplantation or research. Associated with such container(s) can bevarious written materials such as instructions for use, or a notice inthe form prescribed by a governmental agency regulating the labelling,manufacture, use or sale of such products, which notice reflectsapproval by the agency of manufacture, use, or sale for humanadministration. Parts of a kit may be used simultaneously orchronologically staggered, i.e., at different points in time and withequal or different time intervals for any component of a kit. Timeintervals can be selected to obtain the desired effect. The kit mayinclude instructions for temperature of use of each part of the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

Toxicity of CPAs: DMSO, EG, PG, glycerol, formamide

The following methods A and B describe examples for measuring toxicity.

A. Full-thickness articular cartilage samples are stored in Dulbecco'sphosphate buffered saline (PBS) solution (pH=7.0) (Gibco, BRL, MD) at 4°C. Slices of 70 μm thickness are removed using a Vibratome® (TheVibratome Company, St. Louis, Mo.) cutting from the articular surface tothe bone-cartilage junction. The 70 μm thickness is sufficient to allowrapid CPA equilibration throughout the sample.

One control solution of 1× PBS and four experimental solutions of 1M,3M, 5M, and 6M DMSO (Fisher; 99.9% pure; wt/vol) in 1× PBS are prepared.Individual samples are randomly assigned to one of five solutions andone of three temperatures (4° C., 22° C., or 37° C.) with multiplereplicates. Slices are taken from each sample and immersed in 4 ml ofone of the assigned solutions and temperatures in a multi-well tissueculture plate (Corning Inc., Corning, N.Y.) for various lengths of time(ranging from 0.5 min to 120 min).

Once the appropriate experimental time has elapsed, the slices areremoved from their respective solution and rinsed with 1×PBS. Each sliceis stained using membrane integrity dyes of Syto 13 (Molecular Probes,Eugene, Oreg.) and ethidium bromide (EB; Sigma, St. Louis, Mo.) [0.1% EBwith 0.45% Syto mixed in PBS (vol/vol)] and viewed under a Leitz Dialux22 fluorescence (440-480 nm) microscope (Leica Microsystems, RichmondHill, ON) at 125× magnification (10× objective and 12.5× eyepiece). Tworepresentative images from each sample are recorded by digital camera(Pixera DiRactor, Pixera Corporation, Los Gatos, CA) and stored oncomputer for later analysis. The images are then analyzed with a customcell counting program (Viability 3.1, The Great Canadina ComputerCompany, Spruce Grove AB) that utilizes minimum pixel intensity toapproximate the numbers of green (intact) cells and red (disrupted)cells. The cell viability (ratio of intact cells to total cells) inindividual slices is normalized against the cell viability or the totalnumber of cells in control slices maintained in 1×PBS. This is repeatedin at least triplicate.

In order to confirm the results of the membrane integrity assay, asecond viability assay is utilized. The assay measures the reduction ofthe tetrazolium salt WST-1 by mitochondrial dehydrogenases in viablecells. After exposure to the control solution of 1×PBS or the fourexperimental solutions of 1M, 3M, 5M and 6M DMSO, 10 slices are randomlyimmersed in 2 ml of one of the five solutions in a 24-well tissueculture plate (Costar #3526, Corning Inc., Corning, N.Y.) for variouslengths of time (ranging from 0 min to 120 min) at 22° C. Once theappropriate experimental time has elapsed, the slices of AC are removedfrom their respective solution and immersed in 2 ml of 1×PBS in another24-well tissue culture plate for 10 minutes to allow for CPA removal.The slices are then placed in another 24-well plate with each wellcontaining 200 μl of appropriate growth media and 30 μl of WST-1 cellproliferation reagent (Roche Diagnostics, Indianapolis, Ind.). Theplates are incubated at 37° C. in 5% CO₂ for 24 hours. A 100 μl aliquotof media from each well is transferred to a 96-well flat-bottom plate(Costar #3595, Corning Inc., Corning, N.Y.) and absorbency is measuredat 450 nm using a SpectraMax Plus 384™ microplate spectrophotometer(Molecular Devices, Sunnyvale, Calif.).

Media and WST-1 reagent are incubated in parallel with samples. Theresultant spectrophotometer readings are subtracted from readings fortreated samples to eliminate background effects. Spectrophotometerreadings are normalized against pooled control samples incubated in1×PBS over the same period. All samples are repeated at least intriplicate.

B. Cells from the tissue samples are isolated by digesting thesurrounding matrix. For example, for articular cartilage, the tissue isplaced in a 100 mm diameter petri dish containing 1× Dulbecco'sphosphate buffered saline solution (pH 7.0) (PBS, Gibco, BRL, MD). ThePBS is removed and 20 mL of Dubelcco's modified eagle media with F-12nutritional supplement and 1% penicillin-streptomycin (DMEM/F-12, Gibco,BRL, MD) containing 1 mg/mL of collagenase 1A (Sigma-Aldrich Canada,Oakville, ON,) is added. The cartilage is incubated with shaking at 37°C. and 5% CO₂ for 6 hours and then the solution is passed through a 40μm nylon cell strainer (BD biosciences, Mississauga, ON). The solutionis centrifuged at 500 rcf for 6 minutes to pellet the chondrocytes; theyare washed once in PBS, and then plated on the first 7 rows of 96-wellclear tissue culture treated microplates (Costar, Corning Inc LifeSciences, Lowell, Mass.) at 25,000 cells per well in 100 μL DMEM/F-12with 10% fetal bovine serum (FBS, Gibco, BRL, MD). After leaving theplates at room temperature for 30 minutes to allow the chondrocytes tosettle they are incubated at 37° C. and 5% CO₂ for 36-48 hours to allowthe chondrocytes to attach to the plates and recover from the stress ofthe collagenase digestion. Half of the supernatant is removed from eachwell using a 96-well pipetting device (vacupette, Bel-Art products,Pequannock, N.J.); 1004 of DMEM/F-12 with 10% FBS (DMEM/F-12/FBS) isadded and removed from each well using a vacupette to wash the cells.100 μL of DMEM/F-12/FBS is added to the positive control wells and tothe blank wells (FIG. 1) and 100 μL of pure dimethyl sulfoxide (DMSO,Fisher, Fair lawn, N.J.) is added to the negative control wells.

Increasing concentrations of each CPA are added to individual wellsuntil the desired concentration is reached. Each addition is calculatedto limit osmotic expansion/contraction of the cell to less than 40% ofits original volume to prevent overexpansion/contraction cell lysis.After each addition, three minutes are allowed to pass to permit fullequilibration prior to the next higher concentration of that solution.

After the desired concentration of CPA is achieved, the plates areincubated at 37° C. and 5% CO₂ for one of 5, 20, 60 or 120 minutes. Atthe end of the incubation 100 μL of solution is removed from all wellsand replaced with 100 μL of DMEM/F-12/FBS; this solution is allowed toequilibrate for 3 minutes then 100 μL of solution is again removed andreplaced to achieve a slower dilution of CPA and prevent osmotic inducedcell damage. This solution is allowed to equilibrate for 3 minutes; thenthe wells are emptied. 100 μL of WST-1 (Roche diagnostics, Laval, QC) insolution (10 μL WST-1:100 μL phenol red free DMEM/F-12/FBS) is added toeach well of the plate and the plate is incubated at 37° C. and 5% CO₂for 90 minutes. The absorbance of the solutions in each well is measuredusing a spectrophotometer (SpectraMax Plus 384, Molecular devices,Sunnyvale, Calif.) reading at 450 nm using 650 nm as a backgroundreference. The wells are again emptied and 40 μL of SYTO 13 (Invitrogen,Eugene, Oreg.)(1.7%)/ethidium bromide (0.34%) is added and leftprotected from light for 30 minutes. The fluorescence of each well ismeasured using a fluorometer (SpectraMax Gemini EM, Molecular devices,Sunnyvale, Calif.) (excitation 460/emission 510(Syto13); excitation490/emission 610 (EB)). Images from representative wells are capturedusing a 40 times magnification inverted fluorescent microscope (EclipseTE 2000-U, Nikon Canada Inc, Mississauga, ON), the images analysed usinga custom cell counting program (Viability 3.1, The Great CanadianComputer Company, Spruce Grove, AB) and used to standardise cell numberand viability readings from the fluorometric data.

Permeation Kinetics of CPAs in AC matrix

Full thickness AC (on 5-10 mm bone base) is harvested from the sampletissue using a hand-held coring device and held at 4° C. in 1×Dulbecco's phosphate-buffered saline (PBS) solution (pH 7.1) (GibcoInvitrogen, Carlsbad, Calif.). Each osteochondral dowel (OCD) isassigned to one of four CPA treatment groups [DMSO, PG, EG, glycerol(all at 6.5M, in 1× PBS)] and one of 11 incubation times (1 second, 1,2, 5, 10, 15, 30, 60, 120, 180 minutes, 24 hours), as well as one ofthree temperatures (4, 22, 37° C.). Each combination of treatment, time,and temperature is repeated at least three times.

The tissue sample is isolated by scalpel and the tissue returned to 1×PBS for 5 min, then blotted lightly using Kimwipes® tissue(Kimberly-Clark, Roswell, Ga.) to remove excess fluid from the samplesurface, weighed (W1), and immediately immersed in 5 ml of CPA solutionfor the specified incubation time at the specified temperature. Afterincubation, the sample is removed from the solution, blotted lightly andweighed for a second time (W2). The sample is then placed into a 35×10mm cell culture dish (Corning Inc., Corning, N.Y.) containing 4 mL of 1×PBS, fully immersed in the PBS and the dish sealed with Parafilm®(American National Can, Chicago, Ill.) and held for 24 h at 22° C. underdark conditions to allow the CPA within the AC disc to fully equilibratewith the surrounding PBS solution. After 24 h, the CPA/PBS solution ismixed using a pipettor and a 1 mL sample is taken into a 1.5 mLmicrocentrifuge tube (Thermo Fisher Scientific, Waltham, Mass.). Fromthis sample, 50 μL is placed into a μOSMETTE™ micro-osmometer (PrecisionSystems, Natick, Mass.) to determine its osmolality (Osm).

Calculations

As the measured osmolalities are quite low, the immersion solution canbe considered ideal and dilute.

The number of moles of CPA in the surrounding solution is calculated as:

$\begin{matrix}{{n_{s}({mole})} = \frac{\left( {\pi_{s} - \pi_{PBS}} \right)\left( {{mosm}\text{/}{kg}} \right) \times 4\mspace{14mu} {mL} \times 0.99770\; \left( {g\text{/}{mL}} \right)}{1000\left( {{mosm}\text{/}{osm}} \right) \times 1000\left( {g\text{/}{kg}} \right)}} & (1)\end{matrix}$

where π_(s)=osmolality of the final solution, π_(PBS)=osmolality of theinitial PBS solution, and 0.99770=density of water at 22° C.

The total number of moles of CPA that had permeated into the sample isthen given by:

n _(total)(mole)n _(s)(mole)+n _(inside cartilage)(mole)   (2)

-   -   Note: n_(inside cartilage) was estimated to be negligible and        has been omitted from the calculations.

The weight of the CPA is calculated as:

$\begin{matrix}{{{Wt}_{CPA}(g)} = {{n_{total}({mole})} \times {{MW}_{CPA}\left( \frac{g}{mole} \right)}}} & (3)\end{matrix}$

where MW_(CPA) is the molar mass of the CPA.

The volume of CPA was then calculated as:

$\begin{matrix}{{V_{CPA}({mL})} = \frac{{Wt}_{CPA}(g)}{{Density}_{CPA}\left( \frac{g}{mL} \right)}} & (4)\end{matrix}$

Note: True (pure) densities for each CPA at corresponding temperaturesmay be obtained from the literature or a commercial simulation packagesuch as Aspen-HYSYS v. 2004.2.

The amount of water within isotonic cartilage is measured to be77.6±0.5% (S.E.) by mass in a previous study. Assuming a constant dryweight percentage of 22.4%, the dry weight of each AC disc is calculatedas:

Dry Weight(g)=W1(g)×0.224   (5)

The volume of water in the sample after 24 h equilibration in 1× PBS iscalculated as:

$\begin{matrix}{{V_{{water}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cartilage}}({mL})} = \frac{{W\; 2(g)} - \left\lbrack {{{Dry}\mspace{14mu} {{Weight}(g)}} + {{Wt}_{CPA}(g)}} \right\rbrack}{1\left( \frac{g}{mL} \right)}} & (6)\end{matrix}$

Finally, the concentration of CPA that penetrated the AC disc wascalculated to be:

$\begin{matrix}{{\lbrack{CPA}\rbrack \left( \frac{mol}{L} \right)} = \frac{{n_{total}({mole})} \times 1000\left( \frac{ml}{L} \right)}{{V_{CPA}({mL})} + {V_{{water}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cartilage}}({mL})}}} & (7)\end{matrix}$

Note that this is the solution concentration (i.e. moles per fluidvolume in sample and not moles per sample volume).

Exposure Time Determination

The exposure time in each experiment was determined based on thephysical understanding of the CPA diffusion in cartilage gained from thetriphasic model by Abazari et al. (A biomechanical triphasic approach tothe transport of nondilute solutions in articular cartilage. Author(s):Abazari A, Elliott JAW, Law G K, McGann L E, Jomha N M. Source:BIOPHYSICAL JOURNAL Volume: 97 Issue: 12 Pages: 3054-3064 Published:Dec. 16 2009):

That there exists a time-dependent spatial distribution of the CPAwithin the cartilage during CPA diffusion.

That the minimum CPA concentration in this setup (i.e. forcryopreservation of cartilage on the bone) is always at thebone-cartilage interface.

That using the average value for the CPA concentration in cartilage asthe target for permeation can result in partial loss of the cells due tofreezing of half of the cartilage near the bone due to inadequateconcentration of the CPA there, and that the diffusion time of the CPAin each step of the experiments is preferably determined such that theminimum concentration, not the average concentration, reaches therequired concentration for each step.

That Fick's law of diffusion always overestimates the diffusion time ofthe CPA in cartilage compared to the biomechanical model. Therefore,using Fick's law for the calculation of the diffusion times ensures thatthe minimum required concentration in the cartilage is reached.

Based on the above-mentioned understandings, permeation times of the CPAin cartilage were calculated using predictions of Fick's law for theone-dimensional diffusion of each CPA in cartilage:

$\begin{matrix}{\frac{\partial C_{CPA}}{\partial t} = {{- D}\frac{\partial^{2}C_{CPA}}{\partial x^{2}}}} & (8)\end{matrix}$

The values of the diffusion coefficients for the single CPA diffusion inwater were used in the calculations. The values of the diffusioncoefficients were calculated before, by fitting Fick's law to theexperimental data, as explained in the preceding section of this patentlabeled “Permeation Kinetics of CPAs in AC matrix”.

The initial concentration was set to zero, i.e., C_(CPA)(x, t=0)=0, andthe average initial thickness was THK=2 mm.

The boundary conditions, for the cartilage on bone, were

${\frac{\partial C_{CPA}}{\partial x} = {{0\mspace{14mu} {at}\mspace{14mu} x} = 0}},$

and C_(CPA)=C* at x=THK, where C* is the concentration of the CPA in theexternal bath at each step of the protocol. The temperature dependenceof the diffusion coefficients was calculated by fitting an Arrheniusequation to the values of diffusion coefficients obtained for each CPAat 3 different temperatures as in the preceding section of this patentlabeled “Permeation Kinetics of CPAs in AC matrix”. The activationenergies, Ea, for the 4 CPAs (D, EG, PG and G) are tabulated in Table(1). The values of diffusion coefficients were taken from Jomha et al (NM Jomha, G K Law, A Abazari, K Rekieh, JAW Elliott, LE McGann.Permeation of several cryoprotectant agents into porcine articularcartilage. Cryobiology 58(1), 110-114, 2009). For F (formamide), 2values for diffusion coefficients of formamide in water at 2 differenttemperatures were extracted from the literature:

Diffusion coefficient Temperature (K) (m²/s) Reference 278.2 0.95 × 10⁻⁹[1] 298.15 1.58 × 10⁻⁹ [2] [1] Albright JG, Gosting LJ. The diffusioncoefficient of formamide in dilute aqueous solutions at 25° as measuredwith the gouy diffusiometer J. Phys. Chem., 1960, 64 (10), pp 1537-1539.[2] Easteal AJ, Woolf LA. Pressure and temperature dependence of tracerdiffusion coefficients of methanol, ethanol, acetonitrile, and formamidein water, J. Phys. Chem., 1985, 89 (7), pp 1066-1069)

Then, these values were fitted to an Arrhenius equation to calculate theconstant and activation energy for formamide. Those values are alsotabulated in Table (1). As an example, the increase in the minimum DMSOconcentration in cartilage, diffusing from a bath of 3 M DMSO solution,at −10° C., was calculated by solving Eqn. (8) as the following:

First, the diffusion coefficient of DMSO in water at −10° C. wascalculated from the results of the Arrhenius fit in Table (1).

D _(DMSO)(at T=−10° C.=263 K)=298.95×10⁻⁹×exp(−3.9/0.001986/263)=1.71×10⁻¹⁰ m²/s

Eqn. (8) was solved using COMSOL Multiphysics® with initial and boundaryconditions as previously mentioned, and the value of minimumconcentration, i.e. C_(CPA) at x=0, was plotted versus time as in FIG.2).

From FIG. 2, the minimum concentration of DMSO in cartilage can be foundat any time. Therefore, for example, if a minimum concentration of 1.2 Mis desired in one step at −10° C. with DMSO concentration of 3 M in theexternal bath, the cartilage must be immersed in the bath for 120minutes.

TABLE (1) The activation energies and constants obtained for 5 CPAs byfitting the results from the preceding section of this patent labeled“Permeation Kinetics of CPAs in AC matrix” and literature data with anArrhenius equation.$D_{cw} = {D_{cwo} \times {\exp \left( {- \frac{E_{a}}{RT}} \right)}}$D (DMSO) D_(cwo) (× 10⁻⁹ m²/S) 298.95 E_(a) (Kcal/mol) 3.9 E (EG)D_(cwo) (× 10⁻⁹ m²/S) 183.30 E_(a) (Kcal/mol) 3.8 P (PG) D_(cwo) (× 10⁻⁹m²/S) 16971.0 E_(a) (Kcal/mol) 6.63 G (Gly) D_(cwo) (× 10⁻⁹ m²/S) 208.03E_(a) (Kcal/mol) 5.6 F (Form) D_(cwo) (× 10⁻⁹ m²/S) 1903.510 E_(a)(Kcal/mol) 4.198 R = 0.001986 Kcal/(mol.K), T in degrees K

EXAMPLE 1 Vitrification Protocol

A full-thickness articular cartilage dowel was removed from the distalend of the femur and placed in XVIVO at 4° C. with 0.1 mg/mL chondroitinsulphate. All subsequent solutions contained 0.1 mg/mL chondroitinsulphate. The dowel was then placed in 40 mL of 6M dimethyl sulphoxide(DMSO, pre-cooled to 0° C.) and incubated for 1 hr 30 min to achieve a“minimum” concentration of 2.438M DMSO within the cartilage matrix. Thedowel was then removed, quickly blotted, and incubated in a solutioncontaining 2.4375M DMSO and 6M glycerol (pre-cooled to 0° C.) for 3 hrand 40 min. to achieve minimum concentrations of 2.4375M DMSO and 1.625Mglycerol within the matrix. Again, the dowel was removed, blotted, andincubated in a solution (pre-cooled to −10° C.) containing 2.4375M DMSO,1.625M glycerol, and 6M propylene glycol (PG) for 3 hr and 3 min. toachieve minimum concentrations of 2.4375M DMSO, 1.625M glycerol, and0.8125M PG. The dowel was removed, blotted, and incubated in a solution(pre-cooled to −15° C.) containing 2.4375M DMSO, 1.625M glycerol,0.8125M PG and 6M EG for 1 hr and 20 min giving final matrix minimumconcentrations of 2.4375M DMSO, 1.625M glycerol, 0.8125M PG and 1.625MEG. The dowel in this solution was then plunged into liquid nitrogen for20 min to achieve vitrification of the cryoprotectant solution andcryopreservation of the tissue. Another sample was held in liquidnitrogen for 3 months. To evaluate the resultant effectiveness of thisvitrification procedure, the tube was re-warmed in a water bath at 37°C. until the dowel could be removed. The dowel was then placed in 150 mLof XVIVO for 30 min at 4° C., with gentle agitation to remove the CPAsfrom the cartilage matrix. Full-thickness slices were then taken andstained with fluorescent dyes SYTO-13/Ethidium bromide and cell membraneintegrity was assessed.

All solutions were made in XVIVO and contained 0.1 mg/mL chondroitinsulphate. All times were pre-determined based on permeation kinetics foreach CPA using 2 mm as the estimated cartilage thickness. FIG. 1 is aflow-chart illustrating one embodiment on how the method used in thisexample can be used to vitrify the AC for subsequent transplantation.

In one example, of a method, a full thickness (approximately 2 mm thick)10 mm diameter dowel of human articular cartilage was permeated,vitrified and warmed using the described solution: (1) 2.4375 DMSO, (2)1.625M glycerol, (3) 0.8125M PG and (4) 1.625M EG with each CPA added atprogressively lower temperatures. Subsequent staining with Syto 13 andethidium bromide highlighted intact and disrupted cells. Manual countingof cells recorded approximately 77% intact cell recovery in this humantissue after the vitrification procedure repeated on nine different kneearthroplasty samples held for 20 min in liquid nitrogen (Table 2k) andone human cadaveric donor (3 repeats) resulted in 83% recovery (Table21). In the sample held for 3 months, the cell recovery wasapproximately 73%.

EXAMPLE 2 Vitrification Protocol

A full-thickness articular cartilage dowel was removed from the distalend of the femur and placed in XVIVO at 4° C. without chondroitinsulphate. The permeation times were calculated to provide a “minimum”concentration within the matrix. The same procedure as above wasfollowed with the following solution exposures:

-   -   6M glycerol for 3 hr and 40 min at 0° C.,    -   1.625M glycerol and 6M DMSO for 1 hr and 27 min at 0° C.,    -   1.625M glycerol, 1.625M DMSO and 6M formamide for 50 min at −10°        C.,    -   and 1.625M gly, 1.625M DMSO, 1.625M formamide and 6M EG for 1 hr        18 min at −15° C. to give a final concentration of 1.625M        glycerol, 1.625M DMSO, 1.625M formamide, and 1.625M EG within        the cartilage matrix. All solutions were made in XVIVO. The test        tube was then plunged into liquid nitrogen for 20 min to achieve        vitrification of the cryoprotectant solution and        cryopreservation of the sample. The samples were warmed as        above. The cell recovery determined by membrane integrity stains        was approximately 60% based on two independent samples (Table        2q).

The same procedure was completed with permeation times calculated toprovide an “average” concentration within the matrix resulting in thefollowing procedure:

-   -   6M glycerol for 3 hr at 0° C.,    -   1.625M glycerol and 6M DMSO for 2 hr at 0° C.,    -   1.625M glycerol, 1.625M DMSO and 6M formamide for 1 hr at 0° C.,    -   and 1.625M gly, 1.625M DMSO, 1.625M formamide and 6M EG for 1 hr        30 min at −10° C. to give a final concentration of 1.625M        glycerol, 1.625M DMSO, 1.625M formamide, and 1.625M EG within        the cartilage matrix. The test tube was then plunged into liquid        nitrogen for 20 min to achieve vitrification of the        cryoprotectant solution and cryopreservation of the sample. The        samples were warmed as above. The cell recovery determined by        membrane integrity stains was approximately 44% based on three        samples (Table 2p).

EXAMPLE 3 Vitrification Protocol

A full-thickness articular cartilage dowel was removed from the distalend of the femur and placed in XVIVO at 4° C. with 0.1 mg/mL chondroitinsulphate. The same procedure as above was followed with the followingsolution exposures:

-   -   6M glycerol for 3 hr 40 min at 0° C.,    -   1.625M glycerol and 6M EG for 2 hr 32 min at 0° C.,    -   1.625M glycerol, 2.4375M EG and 6M DMSO for 1 hr 42 min at −10°        C.,    -   1.625M gly, 2.4375M EG, 1.625M DMSO and 6M form for 53 min at        −15° C. to give a final concentration of 1.625M gly, 2.4375M EG,        1.625M DMSO and 0.8125M form within the cartilage matrix. The        test tube was then plunged into liquid nitrogen for 20 min to        achieve vitrification of the cryoprotectant solution and        cryopreservation of the sample. The samples were warmed as        above. The cell recovery determined by membrane integrity stains        was approximately 48% based on seven independent samples (Table        2u).

EXAMPLE 4 Sample Vitrification Protocols and Results

The following tables demonstrate a variety of different combinations ofCPAs using different numbers of CPAs, in varying order and withdifferent exposure times and different concentration end points (i.e.“minimum” or “average” permeation concentrations). Chondroitin sulphate(0.1 mg/mL) was added to only those solutions where stated. In thosestated protocols, the chondroitin sulphate was added to all solutionsused with the exception of the initial solution following cartilageharvest in the operating room. The following tables combine dataobtained from human cadaveric donors (denoted by “CTC”) and cartilageremoved from knee joints during total knee arthroplasty surgicalprocedures (denoted by “TKA”). In addition UAH denotes a sample from theUniversity of Alberta Hospital while M is denotes a sample from theMisericordia Hospital (Edmonton).

TABLE 2a D-G-PG (8M): Permeation times calculated for “average”concentration throughout the matrix. 6M DMSO for 40 min at 0° C. 3MDMSO, 6M gly for 65 min at −7° C. 3M DMSO, 2M gly, 6M PG for 4 h at −15°C. Final concentration = 3M DMSO, 2M gly, 3M PG. Treatment LocationRecovery (%) Age Sex Source Date Trial D-G-PG (8M) Femoral 43 19 FemaleCTC Oct. 2, 2007 45 donor Tibial 14 19 Female CTC Oct. 2, 2007 + 1 45donor Femoral 4.97 76 Female TKA14 Jan. 28, 2010 65 Femoral 8.91 69 MaleTKA15 Jan. 28, 2010 65 Femoral 15.27 79 Female UAH16 Jan. 28, 2010 65Avg 17.23

TABLE 2b G-EG-D (8M): Permeation times calculated for “minimum”concentration throughout the matrix. 6M gly for 5 h at 0° C. 2.67M gly,6M EG for 2 h at 0° C. 2.67M gly, 2.67M EG, 6M DMSO for 2 h at −10° C.Final concentration = 2.67M gly, 2.67M EG, 2.67M DMSO. TreatmentLocation Recovery (%) Age Sex Source Date Trial G-EG-D (8M) Femoral 2849 Male CTC Sep. 2, 2009 61 donor Femoral 17.98 60 Female TKA9 Jan. 13,2010 65 Femoral 1.66 59 Female TKA12 Jan. 15, 2010 65 Femoral 15.65 77Female TKA13 Jan. 15, 2010 65 Avg 15.82

TABLE 2c G-D-F (8M): Permeation times calculated for “minimum”concentration throughout the matrix. 6M gly for 4 h at 0° C. 2.67M gly,6M DMSO for 2 h at 0° C. 2.67M gly, 2.67M DMSO, 6M F for 2 h at −10° C.Final concentration = 2.67M gly, 2.67M DMSO, 2.67M F. Treatment LocationRecovery (%) Age Sex Source Date Trial G-D-F (8M) Femoral 36.12 76Female TKA14 Jan. 28, 2010 65

TABLE 2d G-EG-F (8M): Permeation times calculated for “minimum”concentration throughout the matrix. 6M gly for 4 h at 0° C. 2.67M gly,6M EG for 2 h at 0° C. 2.67M gly, 2.67M EG, 6M formamide for 2 h at −10°C. Final concentration = 2.67M gly, 2.67M EG, 2.67M F. TreatmentLocation Recovery (%) Age Sex Source Date Trial G-EG-F (8M) Femoral 3317 Male CTC31 Nov. 5, 2009 x Femoral 41 17 Male CTC31 Nov. 5, 2009 xFemoral 0.25 49 Male CTC1 Dec. 9, 2009 65 Femoral 0.8 49 Male CTC1 Dec.9, 2009 65 Femoral 1.44 59 Female TKA11 Jan. 15, 2010 65 Femoral 2.08 59Female TKA12 Jan. 15, 2010 65 Femoral 0.68 77 Female TKA13 Jan. 15, 201065 Avg 11.32

TABLE 2e D-G-PG-EG (8M): Permeation times calculated for “average”concentration throughout the matrix. 6M DMSO for 40 min at 0° C. 3MDMSO, 6M gly for 65 min at −7° C. 3M DMSO, 2M gly, 6M PG for 30 min at−15° C. 3M DMSO, 2M gly, 1M PG, 6M EG for 35 min at −15° C. 3M DMSO, 2Mgly, 1M PG, 2M EG for 2 h at −15° C. Final concentration = 3M DMSO, 2Mgly, 1M PG, 2M EG. Treatment Location Recovery (%) Age Sex Source DateTrial D-G-PG-EG (8M) Femoral 52 19 Female CTC Oct. 2, 2007 45 donorFemoral 30 19 Female CTC Oct. 2, 2007 45 donor Femoral 27 17 Male CTC31Nov. 5, 2009 x Femoral 33 17 Male CTC31 Nov. 5, 2009 x Femoral 3 49 MaleCTC1 Dec. 9, 2009 65 Femoral 3 49 Male CTC1 Dec. 9, 2009 65 Femoral38.76 64 Female TKA1 Nov. 26, 2009 65 Femoral 24.48 76 Female TKA2 Nov.26, 2009 65 Femoral 11.23 68 Male TKA3 Nov. 26, 2009 65 Avg 24.7

TABLE 2f G-EG-F-D-PG: Permeation times calculated for “minimum”concentration within matrix for gly and EG and “average” concentrationthroughout the matrix for F, DMSO, and PG. 6M gly for 4 h at 0° C. 2.67Mgly, 6M EG for 3 h at 0° C. 2M gly, 2M EG, 6M formamide for 30 min at−10° C. 2M gly, 2M EG, 1.5M formamide, 6M DMSO for 30 min at −10° C. 2Mgly, 2M EG, 1.5M formamide, 1.25M DMSO, 6M PG for 30 min at −10° C.Final concentration = 2M gly, 2M EG, 1.5M formamide, 1.25M DMSO, 1.25MPG. Treatment Location Recovery (%) Age Sex Source Date TrialG-EG-F-DG-P Femoral 2 17 Male CTC31 Nov. 5, 2009 x

TABLE 2g G-EG-F-D-PG-Me-Et: Permeation times calculated for “minimum”concentration within the matrix for G, EG, F, and methanol and “average”concentration throughout the matrix for DMSO, ethanol and PG 6M gly for4 h at 0° C. 2.67M gly, 6M EG for 3 h at 0° C. 2.67M gly, 2.67M EG, 6Mformamide for 20 min at −10° C. 2M gly, 2M EG, 0.4M formamide, 6M DMSOfor 20 min at −10° C. 2M gly, 2M EG, 0.4M formamide, 0.4M DMSO, 6M PGfor 20 min at −10° C. 2M gly, 2M EG, 0.4M formamide, 0.4M DMSO, 0.4M PG,6M methanol for 20 min at −10° C. 2M gly, 2M EG, 0.4M formamide, 0.4MDMSO, 0.4M PG, 0.4M methanol, 6M ethanol for 20 min at −10° C. Finalconcentration = 2M gly, 2M EG, 0.4M formamide, 0.4M DMSO, 0.5M PG, 0.4Mmethanol, 2.3M ethanol. Treatment Location Recovery (%) Age Sex SourceDate Trial G-EG-F-D-PG-Me-Et Femoral 28 17 Male CTC31 Nov. 5, 2009 xFemoral 74.36 52 Female TKA8 Jan. 13, 2010 65 Femoral 11.99 60 FemaleTKA9 Jan. 13, 2010 65 Femoral 33.52 76 Female TKA14 Jan. 28, 2010 65Femoral 4.98 69 Male TKA15 Jan. 28, 2010 65 Femoral 3.16 79 Female UAH16Jan. 28, 2010 65 Avg 26

TABLE 2h G-EG-F-D-PG-Me-Et: Permeation times calculated for “minimum”concentration within the matrix for G, EG, F, methanol and “average”concentration throughout the matrix for DMSO, PG, and ethanol. 6M glyfor 4 h at 0° C. 2.67M gly, 6M EG for 3 h at 0° C. 1.5M gly, 1.5M EG, 6Mformamide for 20 min at −10° C. 1.5M gly, 1.5M EG, 0.8M formamide, 6MDMSO for 20 min at −10° C. 1.5M gly, 1.5M EG, 0.8M formamide, 0.8M DMSO,6M PG for 20 min at −10° C. 1.5M gly, 1.5M EG, 0.8M formamide, 0.8MDMSO, 0.8M PG, 6M methanol for 20 min at −10° C. 1.5M gly, 1.5M EG, 0.8Mformamide, 0.8M DMSO, 0.8M PG, 0.8M methanol, 6M ethanol for 20 min at−10° C. Final concentration = 1.5M gly, 1.5M EG, 0.8M formamide, 0.8MDMSO, 0.62M PG, 0.8M methanol, 2.3M ethanol. Treatment Location Recovery(%) Age Sex Source Date Trial G-EG-F-D-PG-Me-Et Femoral 5.24 52 FemaleTKA8 Jan. 13, 2010 65 Femoral 14.45 60 Female TKA9 Jan. 13, 2010 65 Avg9.85

Further experimentation pursued specific combinations of CPAs includinga 4 component solution consisting of DMSO (D), Glycerol (G), propyleneglycol (PG) and ethylene glycol (EG) in a 3:2:1:2 ratio for a totalconcentration of 6.5M. The permeation times were based on an “average”concentration within the matrix and included:

TABLE 2i −6M DMSO for 30 min at 0° C. −2.4375M DMSO and 6M gly for 45min at 0° C. −2.4375M DMSO, 1.625M gly and 6M PG for 20 min at −5° C.−2.4375M DMSO, 1.625M gly, 0.8125M PG and 6M EG for 25 min at −10° C.For a final solution of 2.4375M DMSO, 1.625M gly, 0.8125M PG, and 1.625MEG. Three different samples were trialed with an average of 37%recovery. Chon std sample treatment Sulf recovery avg dev 1 6.5D-G-PG-EG 0 10.8 2a 6.5 D-G-PG-EG 0 25.9 2b 6.5 D-G-PG-EG 0 74.1 36.933.1

This was repeated using exposure times to reach a calculated “minimum”concentration of each CPA within the matrix. The process included:

-   -   6M DMSO for 1 hr 30 min at 0° C.    -   2.4375M DMSO and 6M gly for 3 h 40 min at 0° C.    -   2.4375M DMSO, 1.625M gly and 6M PG for 3 h 3 min at −10° C.    -   2.4375M DMSO, 1.625M gly, 0.8125M PG and 6M EG for 1 h 20 min at        −15° C.

The final solution consisted of 2.4375M DMSO, 1.625M gly, 0.8125M PG,and 1.625M EG. There was approximately 49% cell recovery in 7 samplesfrom 5 different patients.

TABLE 2j Chon std sample treatment Sulf recovery avg dev 1 6.5 D-G-PG-EG0 45.2 2 6.5 D-G-PG-EG 0 47.4 3a 6.5 D-G-PG-EG 0 73.9 3b 6.5 D-G-PG-EG 061.8 4a 6.5 D-G-PG-EG 0 38.7 5 6.5 D-G-PG-EG 0 49.2 4b 6.5 D-G-PG-EG 029.4 49.4 14.7

The same process was followed with the addition of 0.1 mg/mL chondroitinsulphate with approximately 77% cell recovery from 14 samples from 8different patients.

TABLE 2k Chon std sample treatment Sulf recovery avg dev 1a 6.5D-G-PG-EG 0.1 88.26 1b 6.5 D-G-PG-EG 0.1 48.15 2 6.5 D-G-PG-EG 0.1 81.043a 6.5 D-G-PG-EG 0.1 89.08 3b 6.5 D-G-PG-EG 0.1 86.14 3c 6.5 D-G-PG-EG0.1 76.9 4a 6.5 D-G-PG-EG 0.1 77.72 4b 6.5 D-G-PG-EG 0.1 81.25 5 6.5D-G-PG-EG 0.1 71.19 6a 6.5 D-G-PG-EG 0.1 81.16 6b 6.5 D-G-PG-EG 0.153.06 7a 6.5 D-G-PG-EG 0.1 86.998 7c 6.5 D-G-PG-EG 0.1 81.18 8 6.5D-G-PG-EG 0.1 74.47 76.9 12.3

All of the following solutions had permeation times calculated based on“minimum” concentrations throughout the matrix unless otherwise stated.

The previous results (Table 2k) were obtained using cartilage discardedduring total knee replacement surgery. We repeated the same experimentwith normal articular cartilage from one young normal donor using 3separate 10 mm diameter cores from the same subject with approximately84% cell recovery.

TABLE 21 Chon std sample treatment Sulf recovery avg dev 4a-CTC 6.5D-G-PG-EG 0.1 76.1 4b-CTC 6.5 D-G-PG-EG 0.1 91.4 4c-CTC 6.5 D-G-PG-EG0.1 84.1 83.9 7.7

A pellet culture was performed to ensure the cells were indeed viableand a pellet did form indicating the ability for these cells to producea matrix and the cells were able to produce glycosaminoglycans (aproduct of normal chondrocytes).

TABLE 2m DNA gag (pg) (μg) gag/DNA 11096 8.28 745.80 6415 7.89 1229.4713436 8.97 667.94

Another method to vitrify cartilage included a re-ordering of the CPAs(without chondroitin sulphate) and included:

-   -   6M EG for 1 h 28 min at 0° C.    -   1.625M EG and 6M gly for 3 h 37 min at 0° C.    -   1.625M EG, 1.625M gly and 6M DMSO for 2 h 0 min at −10° C.    -   1.625M EG, 1.625M gly, 2.4375M DMSO and 6M PG for 3 h 58 min at        −15° C.

Resulting in a final solution of 1.625M EG, 1.625M gly, 2.4375M DMSO and0.8125M PG with a recovery rate of approximately 40%.

TABLE 2n Chon std sample treatment Sulf recovery avg dev 1a 6.5EG-G-D-PG 0 46.98 1b 6.5 EG-G-D-PG 0 48.9 2a 6.5 EG-G-D-PG 0 23.2 4a 6.5EG-G-D-PG 0 38.8 4b 6.5 EG-G-D-PG 0 43.4 40.3 10.3

Another variation was to alter the concentrations of CPA from the3:2:1:2 that was used with D-G-PG-EG (without chondroitin sulphate).Thus the exposures were:

-   -   6M DMSO for 1 h 2 min at 0° C.    -   1.5M DMSO and 6M gly for 4 h 13 min at 0° C.    -   1.5M DMSO, 2M gly and 6M PG for 3 h 37 min at −10° C.    -   1.5M DMSO, 2M gly, 1M PG and 6M EG for 2 h 32 min at −15° C.

For a final solution containing 1.5M DMSO, 2M gly, 1M PG, and 2M EG thatresulted in approximately 46% cell recovery after vitrification andwarming.

TABLE 2o Chon std sample Treatment Sulf recovery avg dev 1a 6.5D-G-PG-EG 0 60.3 1b 6.5 D-G-PG-EG 0 40.2 2 6.5 D-G-PG-EG 0 36.2 3 6.5D-G-PG-EG 0 47.5 46.1 10.6

Another four combination CPA solution included glycerol, DMSO, formamideand EG without chondroitin sulphate. This process used the “average”permeation calculations for the CPA exposure times. This solutionincluded:

-   -   6M glycerol for 3 h at 0° C.    -   1.625M gly and 6M DMSO for 2 h at 0° C.    -   1.625M gly, 1.625M DMSO and 6M formamide for 1 h at 0° C.    -   1.625M gly, 1.625M DMSO, 1.625M form and 6M EG for 1 h 30 min at        −10° C.

The final solution of 1.625M gly, 1.625M DMSO, 1.625M form and 1.625M EGresulted in approximately 44% cell recovery.

TABLE 2p Chon std sample treatment Sulf recovery avg dev 1 6.5 G-D-F-EG0 31.9 2 6.5 G-D-F-EG 0 0.6 3 6.5 G-D-F-EG 0 99.2 43.9 50.4

We then performed the same experiment but using the “minimum” permeationcalculation and the solution included:

-   -   6M glycerol for 3 h 40 min at 0° C.    -   1.625M gly and 6M DMSO for 1 h 27 min at 0° C.    -   1.625M gly, 1.625M DMSO and 6M formamide for 50 min at −10° C.    -   1.625M gly, 1.625M DMSO, 1.625M form and 6M EG for 1 h 18 min at        −15° C.

The final solution of 1.625M gly, 1.625M DMSO, 1.625M form and 1.625M EGresulted in approximately 60% cell recovery.

TABLE 2q Chon std sample treatment Sulf recovery avg dev 1 6.5 G-D-F-EG0 47.8 2 6.5 G-D-F-EG 0 71.4 59.6 16.7

When the same solution had 0. lmg/mL chondroitin sulphate added with thesame exposure times, the results decreased to approximately 22%.

TABLE 2r Chon std sample treatment Sulf recovery avg dev 1a 6.5 G-D-F-EG0 26.2 1b 6.5 G-D-F-EG 0 4.9 2a 6.5 G-D-F-EG 0 12.5 2b 6.5 G-D-F-EG 0 313a 6.5 G-D-F-EG 0 28 3b 6.5 G-D-F-EG 0 32 4 6.5 G-D-F-EG 0 16.6 5 6.5G-D-F-EG 21 21.5 11.9

Another reordering of the relevant CPAs was performed and the solutionincluded:

-   -   6M EG for 1 h 28 min at 0° C.    -   1.625M EG and 6M gly for 3 h 37 min at 0° C.    -   1.625M EG, 1.625M gly and 6M form for 50 min at −10° C.    -   1.625M EG, 1.625M gly, 1.625M form and 6M DMSO for 1 h 42 min at        −15° C.

The final solution of 1.625M EG, 1.625M gly, 1.625M form and 1.625M DMSOresulted in approximately 16% cell recovery after vitrification.

TABLE 2s Chon std sample treatment Sulf recovery avg dev 1 6.5 EG-G-F-D0 6.3 2 6.5 EG-G-F-D 0 33.9 3a 6.5 EG-G-F-D 0 18.1 3b 6.5 EG-G-F-D 0 415.6 13.7

Another reordering resulted in:

-   -   6M EG for 1 h 28 min at 0° C.    -   1.625M EG and 6M gly for 3 h 37 min at 0° C.    -   1.625M EG, 1.625M gly and 6M DMSO for 1 h 32 min at −10° C.    -   1.625M EG, 1.625M gly, 1.625M DMSO and 6M form for 53 min at        −15° C.

The final solution of 1.625M EG, 1.625M gly, 1.625M DMSO and 1.625M formresulted in approximately 22% cell recovery after vitrification.

TABLE 2t Chon std sample treatment Sulf recovery avg dev 1 6.5 EG-G-D-F0 6.8 2 6.5 EG-G-D-F 0 9.9 3a 6.5 EG-G-D-F 0 21.6 3b 6.5 EG-G-D-F 0 32 46.5 EG-G-D-F 0 12.5 5a 6.5 EG-G-D-F 0 32.8 5b 6.5 EG-G-D-F 0 35 21.511.9

One more vitrification protocol using 4 CPAs at a 2:3:2:1 ratio andchondroitin sulphate included:

-   -   6M gly for 3 h 40 min at 0° C.    -   1.625M gly and 6M EG for 2 h 32 min at 0° C.    -   1.625M gly, 2.4375M EG and 6M DMSO for 1 h 42 min at −10° C.    -   1.625M gly, 2.4375M EG, 1.625M DMSO and 6M form for 53 min at        −15° C.

The final solution contained 1.625M gly, 2.4375M EG, 1.625M DMSO and0.8125M form and resulted in approximately 48% cell recovery aftervitrification and warming.

TABLE 2u Chon sample treatment Sulf recovery avg std dev 1 6.5 G-EG-D-F0.1 55.7 2 6.5 G-EG-D-F 0.1 64.3 3 6.5 G-EG-D-F 0.1 59.0 4a 6.5 G-EG-D-F0.1 24.9 4b 6.5 G-EG-D-F 0.1 9.7 4c 6.5 G-EG-D-F 0.1 30.2 5a 6.5G-EG-D-F 0.1 111.8 5b 6.5 G-EG-D-F 0.1 39.2 5c 6.5 G-EG-D-F 0.1 39.5 6a6.5 G-EG-D-F 0.1 58.9 6b 6.5 G-EG-D-F 0.1 61 6c 6.5 G-EG-D-F 0.1 53 7a6.5 G-EG-D-F 0.1 39.5 7b 6.5 G-EG-D-F 0.1 23.8 7c 6.5 G-EG-D-F 0.1 55.148.4 23.9

Another solution consisted of DMSO, gly, EG and formamide withchondroitin sulphate added:

-   -   6M DMSO for 1 h 30 min at 0° C.    -   2.4375M DMSO, 6M gly for 3 h 40 min at 0° C.    -   2.4375M DMSO, 1.625M gly, 6M EG for 1 h 53 min at −10 C    -   2.4375M DMSO, 1.625M gly, 1.625M EG, 6M formamide for 53 min at        −15 C        for a final concentration of 2.4375M DMSO, 1.625M gly, 1.625M        EG, and 0.8125M formamide resulting in 53% cell recovery.

TABLE 2v Treatment Location Recovery (%) Age Sex Source Date TrialD-G-EG-F (6.5M) Femoral 53.2 69 Male Mis15a Oct. 13, 2010 69

One more solution contained DMSO, gly, PG, and EG with the addition of 1mg/mL hyaluronic acid:

-   -   6M DMSO for 1 h 30 min at 0° C.    -   2.4375M DMSO, 6M gly for 3 h 40 min at 0 C.    -   2.4375M DMSO, 1.625M gly, 6M PG for 3 h 3 min at −10 C    -   2.4375M DMSO, 1.625M gly, 0.8125M PG, 6M EG for 1 h 20 min at        −15 C        for a final concentration of 2.4375M DMSO, 1.625M gly, 0.8125M        EG, 1.625M EG resulting in 39% cell recovery.

TABLE 2w Treatment Location Recovery (%) Age Sex Source Date TrialD-G-PG-EG- (6.5M) Femoral 39.2 69 Male Mis15b Oct. 13, 2010 69

Further experimental solutions were examined as described below:

-   -   D-G-PG-EG (6.5M):

Permeation times calculated for “average” concentration throughout thematrix.

-   -   6M DMSO for 33 min at 0 C    -   2.4375M DMSO, 6M gly for 45 min at 0 C    -   2.4375M DMSO, 1.625M gly, 6M PG for 20 min at −5 C    -   2.4375M DMSO, 1.625M gly, 0.8125M PG, 6M EG for 25 min at −10 C        Final concentration=2.4375M DMSO, 1.625M gly, 0.8125M PG, 1.625M        EG.

TABLE 2x In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial D-G-PG-EG (6.5M) Femoral 2.35 62 Male TKA6 Mar. 25, 2010 67Femoral 9.08 56 Male TKA22 Apr. 22, 2010 67 Femoral 19.69 62 Male UAH33dMay 12, 2010 67 Avg 10.37

TABLE 2y In XVIVO solution Treatment Location Recovery (%) Age SexSource Date Trial D-G-PG-EG (6.5M) Femoral 65.12 73 Male TKA17b Apr. 15,2010 67 Femoral 37.59 55 Male TKA27c Apr. 30, 2010 67 Femoral 54.46 57Female TKA31a May 7, 2010 67 Avg 52.39

-   -   D-G-PG-EG (8M):

Permeation times calculated for “average” concentration throughout thematrix.

-   -   6M DMSO for 40 min at 0 C    -   3M DMSO, 6M gly for 1 h 5 min at −7 C    -   3M DMSO, 2M gly, 6M PG for 30 min at −15 C    -   3M DMSO, 2M gly, 1M PG, 6M EG for 1 h 35 min at −15 C        Final concentration =3M DMSO, 3M gly, 1M PG, 2M EG.

TABLE 2z In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial D-G-PG-EG (8M) Femoral 17.02 51 Male TKA1 Mar. 19, 2010 67Femoral 12.46 39 Female TKA2 Mar. 25, 2010 67 Femoral 65.13 61 MaleUAH25 Apr. 29, 2010 67 Femoral 69.61 62 Male UAH33c May 12, 2010 67 Avg41.05

TABLE 2aa In X-VIVO solution Treatment Location Recovery (%) Age SexSource Date Trial D-G-PG-EG (8M) Femoral 11.96 57 Female TKA7 Mar. 31,2010 67 Femoral 80.15 74 Male TKA28 Apr. 30, 2010 67 Femoral 58.38 62Male UAH33g May 12, 2010 67 Avg 50.16

-   -   G-D-F (6.5M):

Permeation times calculated for “minimum” concentration throughout thematrix.

-   -   6M gly for 3 h 30 min at 0° C.    -   2.167M gly, 6M DMSO for 2 h 30 min at 0° C.    -   2.167M gly, 2.167M DMSO, 6M F for 1 h 30 min at −10° C. Final        concentration =2.167M gly, 2.167M DMSO, 2.167M F.

TABLE 2bb In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial G-D-F (6.5M) Femoral 26.16 51 Female TKA14 Apr. 8, 2010 67Femoral 30.80 65 Female TKA18b Apr. 15, 2010 67 Femoral 19.94 62 MaleUAH33e May 12, 2010 67 Avg 25.63

TABLE 2cc In X-VIVO solution Treatment Location Recovery (%) Age SexSource Date G-D-F (6.5M) Femoral 83.39 72 Female TKA12 Apr. 1, 2010Femoral 52.92 58 Male UAH19 Apr. 15, 2010 Femoral 16.09 62 Male UAH33fMay 12, 2010 Avg 50.80

-   -   G-D-F (8M):

Permeation times calculated for “minimum” concentration throughout thematrix.

-   -   6M gly for 4 h at 0° C.    -   2.67M gly, 6M DMSO for 3 h at 0° C.    -   2.67M gly, 2.67M DMSO, 6M F for 2 h at −10° C.        Final concentration=2.67M gly, 2.67M DMSO, 2.67M F.

TABLE 2dd In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial G-D-F (8M) Femoral 5.35 64 Female UAH15 Apr. 8, 2010 67Femoral 8.09 78 Male UAH24 Apr. 29, 2010 67 Femoral 66.29 62 Male UAH33aMay 12, 2010 67 Avg 26.58

TABLE 2ee In X-VIVO solution Treatment Location Recovery (%) Age SexSource Date Trial G-D-F (8M) Femoral 39.34 66 Male TKA13 Apr. 7, 2010 67Femoral 4.96 59 Female TKA21 Apr. 22, 2010 67 Femoral 68.08 67 MaleTKA35b May 14, 2010 67 Avg 37.46

-   -   G-D-F-EG (6.5M):

Permeation times calculated for “minimum” concentration throughout thematrix.

-   -   6M gly for 3 h at 0° C.    -   1.625M gly, 6M DMSO for 2 h at 0° C.    -   1.625M gly, 1.625M DMSO, 6M F for 1 h at 0° C.    -   1.625M gly, 1.625M DMSO, 1.625M F, 6M EG for 1 h 30 min at −10°        C.

Final concentration=1.625M gly, 1.625M DMSO, 1.625M F, 1.625M EG.

TABLE 2ff In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial G-D-F-EG (6.5M) Femoral 86.86 59 Male TKA11 Apr. 1, 2010 67Femoral 21.51 85 Female UAH26a Apr. 29, 2010 67 Femoral 52.70 67 FemaleTKA29c May 6, 2010 67 Avg 53.69

TABLE 2gg In X-VIVO solution Treatment Location Recovery (%) Age SexSource Date Trial G-D-F-EG (6.5M) Femoral 60.77 70 Female TKA4 Mar. 25,2010 67 Femoral 0.93 67 Female TKA23 Apr. 29, 2010 67 Femoral 92.00 85Female UAH32b May 10, 2010 67 Avg 51.23

-   -   G-D-F-EG (8M):

Permeation times calculated for “minimum” concentration throughout thematrix.

-   -   6M gly for 4 h at 0° C.    -   2M gly, 6M DMSO for 3 h at 0° C.    -   2M gly, 2M DMSO, 6M F for 2 h at −10° C.    -   2M gly, 2M DMSO, 2M F, 6M EG for 2 h at −10° C.

Final concentration=2M gly, 2M DMSO, 2M F, 2M EG.

TABLE 2hh In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial G-D-F-EG (8M) Femoral 32.91 59 Male TKA3 Mar. 25, 2010 67Femoral 41.95 64 Female TKA29b May 6, 2010 67 Femoral 25.84 57 FemaleTKA31b May 7, 2010 67 Avg 33.57

TABLE 2ii In X-VIVO solution Treatment Location Recovery (%) Age SexSource Date Trial G-D-F-EG (8M) Femoral 56.48 73 Male TKA17a Apr. 15,2010 67 Femoral 49.09 73 Male TKA17a Apr. 15, 2010 67 Femoral 24.99 85Female UAH32a May 10, 2010 67 Avg 43.52

-   -   G-EG-F-D-PG-Me-Et:

Permeation times calculated for “minimum” concentration within thematrix for G, EG, F and methanol with “average” concentration throughoutthe matrix for DMSO, ethanol and PG.

-   -   6M gly for 3 h 30 min at 0° C.    -   2M gly, 6M EG for 2 h 30 min at 0° C.    -   2M gly, 2M EG, 6M formamide for 20 min at −10° C.    -   2M gly, 2M EG, 0.5M formamide, 6M DMSO for 20 min at −10° C.    -   2M gly, 2M EG, 0.5M formamide, 0.5M DMSO, 6M PG for 20 min at        −10° C.    -   2M gly, 2M EG, 0.5M formamide, 0.5M DMSO, 0.5M PG, 6M methanol        for 20 min at −10° C.    -   2M gly, 2M EG, 0.5M formamide, 0.5M DMSO, 0.5M PG, 0.5M        methanol, 6M ethanol for 20 min at −10° C.        Final concentration=2M gly, 2M EG, 0.5M formamide, 0.54M DMSO,        0.5M PG, 0.5M methanol, 2.3M ethanol.

TABLE 2jj In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial G-EG-F-D-PG-Me-Et Femoral 18.89 62 Male TKA9 Mar. 31, 2010 67Femoral 75.50 65 Female TKA18a Apr. 15, 2010 67 Femoral 15.91 62 MaleUAH33h May 12, 2010 67 Avg 34.30

TABLE 2kk In X-VIVO solution Treatment Location Recovery (%) Age SexSource Date Trial G-EG-F-D-PG-Me-Et Femoral 54.31 77 Male TKA16 Apr. 9,2010 67 Femoral 76.02 85 Female UAH26b Apr. 29, 2010 67 Femoral 30.47 62Male UAH33b May 12, 2010 67 Avg 48.02

-   -   G-EG-F-D-PG-Me-Et :

Permeation times calculated for “minimum” concentration within thematrix for G, EG, F and methanol with “average” concentration throughoutthe matrix for DMSO, ethanol and PG.

-   -   6M gly for 4 h at 0° C.    -   2.46M gly, 6M EG for 3 h at 0° C.    -   2.46M gly, 2.46M EG, 6M formamide for 20 min at −10° C.    -   2.46M gly, 2.46M EG, 0.615M formamide, 6M DMSO for 20 min at        −10° C.    -   2.46M gly, 2.46M EG, 0.615M formamide, 0.615M DMSO, 6M PG for 20        min at −10° C.    -   2.46M gly, 2.46M EG, 0.615M formamide, 0.615M DMSO, 0.615M PG,        6M methanol for 20 min at −10° C.    -   2.46M gly, 2.46M EG, 0.615M formamide, 0.615M DMSO, 0.615M PG,        0.615M methanol, 6M ethanol for 20 min at −10° C.        Final concentration=2.46M gly, 2.46M EG, 0.615M formamide,        0.615M DMSO, 0.615M PG, 0.615M methanol, 2.3M ethanol.

TABLE 2ll In PBS solution Treatment Location Recovery (%) Age Sex SourceDate Trial G-EG-F-D-PG-Me-Et Femoral 94.00 54 Female TKA10 Apr. 1, 201067 Femoral 8.48 74 Female TKA20 Apr. 21, 2010 67 Femoral 54.27 57 FemaleTKA31c May 7, 2010 67 Avg 52.25

TABLE 2mm In X-VIVO Treatment Location Recovery (%) Age Sex Source DateTrial G-EG-F-D-PG-Me-Et (8M) Femoral 25.79 59 Male TKA8 Mar. 31, 2010 67Femoral 69.36 64 Female TKA29a May 6, 2010 67 Femoral 0.52 67 Male TKA36May 14, 2010 67 Avg 31.89

Diversification Assessment

Grading system for visual inspection of solutions undergoingvitrification can be seen in the below table 3:

TABLE 3 Grade Vitrification Quality Devitrification 0 Ice formation Iceformation 1 Partially vitrified Devitrified upon rewarming 2 Mostlyvitrified Devitrified upon rewarming 2.5 Vitrified and crackedDevitrified upon rewarming 3.0 Vitrified and no cracks Devitrified uponrewarming 3.3 Vitrified and cracked Partially devitrified upon rewarming3.5 Vitrified and no cracks Partially devitrified upon rewarming 3.7Vitrified and cracked Some devitrification on edge upon rewarming 4Vitrified and no cracks Some devitrification on edge upon rewarming 4.5Vitrified and cracked Did not devitrify upon rewarming 5 Vitrified andno cracks Did not devitrify upon rewarming

The ability to vitrify is given first priority, followed by the absenceof devitrification. Cracking is taken into consideration, but does notnecessarily indicate that a solution is not an effective one. In oneaspect of the invention, a score of 3.7 or higher is desired for avitrification solution.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications, patents and patent applications referred to herein, orreferenced in such documents are incorporated by reference in theirentirety to the same extent as if each individual publication, patent orpatent application was specifically and individually indicated to beincorporated by reference in its entirety. The citation of any referenceherein is not an admission that such reference is available as prior artto the instant invention.

1. A kit comprising cryopreserving agents (CPAs), at least two of whichCPAs being different CPAs from each other and including a first CPA anda second CPA, the kit having instructions for carrying out a method forcryopreserving articular cartilage using more than one CPA, the methoddescribed in the instructions comprising: permeating a sample ofarticular cartilage with a sequence of at least two different CPAscomprising the first CPA and the second CPA, the second CPA beingpermeated into the sample after permeating the sample with the firstCPA, to form combined CPAs having a concentration distribution withinthe sample; the concentration distribution of the combined CPAs beingselected so that upon cooling of the sample, the combined CPAs vitrifyand cryopreserve the sample and vitrifying the sample by cooling thesample below the glass transition temperature of the permeated sample ofarticular cartilage in a sequence of cooling steps corresponding to thesequential addition of CPAs.
 2. The kit of claim 1 further comprising athird CPA and in which in the method described in the instructions thesequence of at least two different CPAs comprises the third CPApermeated into the sample after permeating the sample with the secondCPA.
 3. The kit of claim 2 further comprising a fourth CPA and in whichin the method described in the instructions the sequence of at least twodifferent CPAs comprises the fourth CPA permeated into the sample afterpermeating the sample with the third CPA.
 4. The kit of claim 1 in whichthe first CPA is selected from the group comprising dimethyl sulfoxide(D), ethylene glycol (EG), propylene glycol (PG), glycerol (G) andformamide (F).
 5. The kit of claim 1 in which the second CPA is selectedfrom the group comprising dimethyl sulfoxide (D), ethylene glycol (EG),propylene glycol (PG), glycerol (G) and formamide (F).
 6. The kit ofclaim 1 in which the third CPA is selected from the group comprisingdimethyl sulfoxide (D), ethylene glycol (EG), propylene glycol (PG),glycerol (G) and formamide (F).
 7. The kit of any claim 1 in which theCPAs are selected from the group comprising dimethyl sulfoxide, ethyleneglycol, propylene glycol, glycerol, formamide, methanol and ethanol. 8.The kit of claim 1 in which the fourth CPA is selected from the groupcomprising dimethyl sulfoxide, ethylene glycol, propylene glycol,glycerol.
 9. The kit of claim 1 in which the kit and sequence of atleast two different CPAs comprises five, six, or seven different CPAs.10.-11. (canceled)
 12. The kit of claim 1 in which the CPAs are chosenfrom the group comprising dimethyl sulfoxide, ethylene glycol, propyleneglycol, glycerol, formamide, methanol and ethanol.
 13. The kit of claim1 in which either the first CPA or the second CPA is glycerol.
 14. Thekit of claim 3 in which the sequence of CPAs is D-G-PG-EG, G-EG-D-F,EG-G-D-PG, EG-G-F-D or D-G-EG-F. 15-18. (canceled)
 19. The kit of claim1 in which in the method described in the instructions either a) one ormore of the CPAs in the sequence of CPAs are combined with chondroitinsulphate or b) one or more of the CPAs in the sequence of CPAs iscombined with hyaluronic acid, or a) and b).
 20. The kit of claim 1 inwhich in the method described in the instructions each succeeding CPA ispermeated into the sample along with each preceding CPA and thepreceding CPA added with each succeeding CPA has a concentrationsubstantially equal to the concentration of the preceding CPA in thesample cartilage. 21.-22. (canceled)
 23. The kit of claim 1 in which inthe method described in the instructions the sequence of CPAs comprisesone of the given examples in Tables 2a-2ll and the kit further comprisesthe CPAs in the given example. 24.-25. (canceled)
 26. A method forcryopreserving articular cartilage using more than one cryopreservingagent, comprising: (i) obtaining an articular cartilage sample; (ii)adding one CPA first at a temperature above the freezing point of thetissue and CPA at the outset for a sufficient period of time to obtain adesired degree of CPA tissue permeation; (iii) moving the tissue toanother solution that contains at least one or more CPAs at atemperature equal to or lower than the temperature in step “(ii)”, buthigher than the freezing point of the solution and tissue at the end ofstep (ii) above, for a sufficient period of time to obtain a desireddegree of CPA tissue permeation; (iv) repeating step (iii) with CPAs ata temperature equal to or lower than previously used but higher than thesolution and tissue freezing point, until a high enough CPAconcentration in the tissue is achieved of all the different CPAs tovitrify the solution and effectively cryopreserve the tissue. 27-28.(canceled)
 29. Articular cartilage (AC) tissue cryopreserved using amethod for cryopreserving AC using more than one cryopreserving agent(CPA), the method comprising: permeating a sample of articular cartilagewith a sequence of at least two different CPAs comprising a first CPAand a second CPA, the second CPA being permeated into the sample afterpermeating the sample with the first CPA, to form combined CPAs having aconcentration distribution within the sample; the concentrationdistribution of the combined CPAs being selected so that upon cooling ofthe sample, the combined CPAs vitrify and cryopreserve the sample; andvitrifying the sample by cooling the sample below the glass transitiontemperature of the permeated sample of articular cartilage in a sequenceof cooling steps corresponding to the sequential addition of CPAs.